Lectins are proteins or glycoproteins, typically of plant or even microbial or animal origin which recognise and attach to specific glycoconjugate structures. Orally administered lectins such as kidney bean (Phaseolus vulgaris) lectin, phytohaemagglutinin (PHA), can be powerful extraneous growth factors for the rat gut, inducing fully reversible, polyamine-dependent, hyperplastic growth of the small intestine (Bardocz et al., 1992). The lectin avidly binds to the brush border and is partially transcytosed into the circulation (Pusztai, 1991). At particular doses, lectins such as PHA damage the gut wall, causing coliform overgrowth in the lumen (Pusztai et al., 1993), increasing the rate of lipid mobilisation and glucose oxidation (Grant et al., 1987) and significantly reducing the fractional rate of protein synthesis in skeletal muscle (Palmer et al., 1987; Bardocz et al., 1992). Thus, lectins such as PHA are generally regarded as nutritional toxins because at high oral doses they induce several losses of body lipids, glycogen and muscle protein (Pusztai, 1991) and possibly death.
Safe, non-toxic threshold levels for the oral administration of lectins in human and other animals are not known.
Mucosal cells are those which make up any mucous membrane (the moist membrane lining many tubular structures). Many are cells which provide a protective layer between the external environment and the internal organs of an animal. Examples of mucosal cells include the epithelial cells of the skin, the mucosal cells of the alimentary canal and the tissue covering the eye. A member of disorders of mucosal cells are known, including conditions where cell division is accelerated, decelerated, where the mucosal cells are damaged, or the protective outer layer such as mucous is missing. Conditions related to abnormal control of mucosal cell proliferation may include skin cancers, psoriasis, irritable bowel disease and mucositis. Mucositis is a painful and debilitating condition in which rapidly growing epithelial cells are damaged and the external mucous layer is removed and/or not replaced sufficiently quickly. Mucositis may result in infection by microorganisms which are present, for example in the mouth or gut. The condition is seen as a major side effect in the treatment of cancer. The incidence and severity of mucositis may increase with increasing rounds of cancer therapy, and may ultimately effect patient treatment compliance and survival.
Agents which damage mucosal (and other) cells include chemotherapeutic agents, radiotherapy, chemicals (organic and inorganic), toxins, acids, alkali, any radiation source and free radicals. Chemotherapy and radiotherapy, used either alone, used together or in combination with surgery are the main therapeutic approaches for the treatment of cancer. Chemotherapy uses a cytotoxic agent to directly damage the DNA of a target cell. If a sufficient dose of the cytotoxic agent is administered to a target tissue i.e. a tumour, DNA mis-repair may result in the accumulation of DNA mutations, lesions and chromosomal aberrations that ultimately result in cell death. Radiotherapy uses radiation to either damage the DNA of a target cell directly, or exploits the potential of ionising radiation to produce free radicals which are able to break DNA strands (Steel, 1996).
The principle by which chemotherapy is used to treat cancer is that a cytotoxic drug is administered to inhibit cell division, which may ultimately lead to cell death. As the cancerous cells are usually growing more rapidly than normal tissue, the expectation is that the cytotoxic drug will kill more cancerous cells than normal cells. Unlike radiotherapy however, the cytotoxic drugs are given in such a manner that they act systemically throughout the body. Serious side effects, such as toxicity to vital tissues including bone marrow and white blood cells may limit the dose of cytotoxic drug that can be administered without killing the patient. In a similar manner, the use of radiation to treat cancer does not discriminate between cancerous and normal tissue. The use of radiotherapy is therefore a compromise in trying to induce most damage to the cancerous cells by targeting the radiation without irreparably destroying normal tissue.
Many cytotoxic drugs have been developed and evaluated for the treatment of cancer. The main principle by which these drugs act is that they interfere or inhibit key steps in the cell division pathway. The major drug classes target either DNA replication, DNA repair, chromosome separation or cytoplasmic division. The vast majority of cytotoxic drugs interfere with the synthesis and replication of DNA. 5-fluorouracil (5-FU) and its related analogs are some of the most commonly used cytotoxic drugs in this class. The activity of 5-FU can also be modulated by the addition of reduced folates such as calcium leucovorin (Isacoff et al, 1994). Other cytoxic drugs that inhibit DNA synthesis and replication are known which target different deoxyribonucleotides used to make DNA e.g. cytarabine. DNA strands containing cytarabine directly inhibit the activity of DNA polymerase (Archimund & Thomas, 1994).
A second major class of cytotoxic drugs are those which induce the breakage of DNA strands directly, or those which inhibit the repair of DNA breaks. Cyclophosphamide is an example of a drug that can break DNA strands directly (Sparano & Wiernik, 1994). A third major class of drugs are those that actually disrupt the assembly and disassembly of tubulin, so inhibiting mitosis and cell division directly. Taxanes such as paclitaxel and docetaxel are drugs which polymerise tubular into stable microtubule bundles. Synthetic vinca-alkaloids such as vinorelbine are spindle poisons which exert their anti-tumour effects by preventing the assembly of tubulin into microtubules (Dieras & Pouillart, 1995).
Current radiotherapy practice uses a range of irradiation sources to treat cancer. The most commonly used sources are X-ray, gamma ray, proton or neutron sources of .alpha. or .beta. emitters. In practice, continuous low dose radiotherapy over several days gives the best changes of discriminating between normal and cancerous tissue. However, this technique is limited to the use of radio-isotopes which are currently only effective with a few tumour types, e.g. thyroid cancer. In the clinical situation, most radiotherapy techniques use high doses of radiation which are focused as a beam at the cancerous tissue. Exposure of normal tissue is reduced, where possible, by the use of lead shielding or by rotating the patient such that normal tissue receives a lower dose than the cancerous tissue. Although this approach can be effective, its use may be limited by cumulative exposures of normal tissue to radiation and the resistance of many tumours to high doses of radiation.
It is well known in the art that single cytotoxic agents or radiation sources may be more suitable for certain cancer types. For example, Cisplatin is widely used for testicular cancer, taxanes are more suitable for breast cancer and 5-FU is widely used for colorectal cancers. However, single agent therapies rarely provide a complete cure for cancer and rates of survival are still low. Some improvements have been made in the use of cytotoxic drug cocktails with the use of multiple drug regimes (Au et al, 1996).
A number of compounds have been evaluated in the art for their ability to sensitise cancer cells to the effects of radiation and chemotherapy (so sparing normal tissue). However, the use of radiosensitisers such as vitamin K mimetics Synkavit and Menadione and protectants such as the sulphidryl containing compounds cysteine, cysteamine and Ethylol have also been disappointing (Denekamp, 1996).
Although chemotherapy and radiotherapy are the most widely used treatments for cancer, the rates of survival are limited due to a number of factors. The key factor is that the cytotoxic drug or radiation does not discriminate between normal and cancerous tissue. In most cases, it is impossible to give a sufficient dose of cytotoxid drug or radiation to reliable kill all cancerous tissue as it would prove fatal to the patient. Common side effects for existing therapy regimes include hair loss, bone marrow suppression, nausea, vomiting and diarrhoea (Paulsen et al 1996). In addition, there are also many instances where the use of radiotherapy, particularly to the pelvic regions has resulted in altered gastrointestinal function (Yeoh et al 1993) and long term damage to the gut which requires surgery (van Halteren et al 1993).
A major breakthrough has been made in the last 10 years with the availability of hematopoetic growth factors. It is now possible to give higher doses of cytotoxic drugs and radiation and then rescue tissues such as bone marrow and white blood cells by the administration of recombinant growth factors such as granulocyte-macrophage colony-stimulating factor (Erkisis et al, 1996). Such an approach has enabled improved prognosis and survival rates to be achieved. Whilst epidermal-specific growth factors such as epidermal growth factor are known, the complex nature of gut growth regulation has made it difficult to develop effective gut "rescue" procedures (Podolsky, 1993), As no such growth factor currently exists for the gut, damage to the gastrointestinal tract by cytotoxic drugs and radiation has now become dose limiting.
The present invention utilises the tissue protecting qualities and the metabolic effects of low doses of lectins to protect and repair biological material damaged by radiotherapy and/or chemotherapy. The present invention is of particular interest because of the noted prophylactic effects of lectin compositions (positive growth factors) before treatments such as radiotherapy and/or chemotherapy.
Diseased and damaged cells, which cannot repair or regenerate sufficiently quickly can cause serious and potentially life-threatening health risks. Also a problem in maintaining normal cell functions are metabolic diseases, such as obesity, hyperglycemia, cardiovascular disease, stroke and gastrointestinal disease, including irritable bowel syndrome, inflammatory bowel disease and coeliac disease.
Metabolic disorders include any disorder which is related to and/or a result of the metabolism of the body, in particular obesity and obesity related disorders such as hyperglycaemia, (type II diabetes), cardiovascular, stroke, gastro-intestinal and gastro-intestinal related conditions. A metabolic disorder may require the control of mucosal cell proliferation, or the control of mucosal cell proliferation may be independent of a metabolic disorder.
It is known in the art that high doses of lectins can be detrimental to the metabolism of an animal. For example, high doses of lectins may interfere with the thymus, cause hypertrophy of the pancreas and coliform overgrowth resulting in poor nutrition and growth. The present invention describes for the first time, the beneficial metabolic effects of orally administering low doses of lectins. Surprisingly, it has been found that administration of low lectin doses results in a reduction in body fat content and this can be used as a treatment for obesity and for non-medically related weight loss.
The use of soya in human food is on the increase and soya proteins often supply the bulk of dietary protein in animal nutrition. Unfortunately, as soya contains a number of antinutrients, mainly lectins and trypsin inhibitors, the efficiency of nutritional utilisation of diets containing soya products is below that expected from chemical composition (Gupta, 1987), particularly when these are fed for long periods (Rackis et al, 1986) and with soya whey containing most of the soya antinutrients (Grant et al., 1986). It is a commonly held view that soya products could be more extensively used in both human and animal diets if their antinutritional effects were reduced.
The antinutrient content of most soya products is generally removed by processing based on various methods of heat-treatments (Liener, 1994). However, most of these are expensive and can lead to losses of essential amino acids and production of toxic by-products. Although cheaper and more efficient heat-processing may eventually be developed, other options for reducing the antinutritional effects of soya products include diet manipulation and the design of new feeding strategies. Rendering soya products and particularly its little-used whey fraction free of the main negative effects of antinutrients could bring considerable economical benefits to the feed industry and animal producers.
A particular lectin to which the present invention relates, for all aspects, is Robinia pseudoacacia (black locust, also RPA). RPA is a leguminous tree species that is common in North America and Central Europe. Robinia pseudoacacia produces lectins in its bark, roots, root nodules, phloem, wood and leaves (Geitle and Ziegler 1980) and in the seeds (Van Damme et al 1995b). Although the precise function of the lectins is unknown, it is believed that the lectins may be used as a store for nitrogen in dormant periods (Yoshida et al 1994). It is also known that the level of lectin within the bark increases during winter. Given the known toxicity of Robinia lectins to mammals, (Nishiguchi et al 1997) it is also possible that the bark lectin may act as a defence mechanism against herbivores such as rabbits during winter.
The bark and seed lectins of Robinia pseudoacacia have been characterised at both the biochemical and molecular levels (Van Damme et al., 1995a, 1995b). Root lectin has also been isolated and characterised (Duverger et al 1997). Robinia pseudoacacia lectins (predominantly seed derived) have had a number of uses, primarily as diagnostic and research tools. The specific carbohydrate binding properties (N-acetyl-D-galactosamine) of RPA have been used to investigate the carbohydrate composition of biological tissue (Raedler et al 1982). The mitogenic properties of RPA have also been used in in vitro studies to investigate the biology of isolated lymphocytes (Sabeur et al 1986, Sharif et al 1978 & 1977). Banach et al (1983) showed that intraperitoneal doses of RPA in mice induced a hepatotoxic effect including a rapid fall in liver glycogen. Pusztai (1993) also showed that RPA bound strongly to the gut wall of the rat and induced pancreatic growth at high doses suggesting that RPA may damage the gut wall, promote coliform overgrowth and induce pancreatitis.
The lectins from the bark of Robinia pseudoacacia can be characterised using a range of gel separation, DNA and protein sequencing technique (Van Damme et al 1995a). Native bark lectin eluated from a column consists of two lectins, RPbAI and RPbAII which co-purify at an apparent molecular weight of 120 kDa. Analysis of the RPbAI on an SDS PAGE gel indicates that it is composed of two subunits, polypeptide a and polypeptide b, with molecular weights of 31.5 and 29 kDa respectively. RPbAI is a tetramer composed of all the possible combinations of the a and b polypeptide monomers. Polypeptides a and b have been further characterised by cDNA cloning and sequencing using standard techniques. The predicted protein sequences of polypeptides a and b are shown in FIG. 1.
RPbAII is a tetramer composed of a single monomer polypeptide (subunit) c of 26 kDa molecular weight. Polypeptide c has been further characterised and the cDNa sequenced and the predicted protein sequence is also shown in FIG. 1.
Robinia pseudoacacia seed contains two lectins, RPsAI and RPsAII which co-purify at an apparent molecular weight of 120 kDa (Van Damme et al., 1995b).
Analysis of both lectins on SDS PAGE indicates that they are each comprised of a single monomer subunit (34 and 29 kDa respectively). The seed lectins have been characterised by cDNA cloning and sequencing techniques and the predicted protein sequence is shown in FIG. 2. Recently published work on the root lectins has shown that they are composed of two lectins (Duverger et al, 1997). The root lectins appear to differ from those of seed and bark in that they are dimer molecules classified as RPrAI and RPrAII of molecular weights 58 and 63 kDa respectively. RPrAI is a heterodimer consisting of a 31 and a 29 kDa subunit and RPrAII is a homodimer consisting of single 30 kDa subunits.
Studies on Robinia pseudoacacia lectins have generated some confusion concerning the precise molecular weight, and subunit composition of the individual lectins. Wantyghem et al (1986) identified two lectins in seeds, RPA 1 a dimer and RPA 3 a tetramer. However, in the same year Fleischmann et al (1986) described the isolation of two tetramer lectins terms RPA 1 and RPA 2 with similar subunit molecular weights. It appears therefore that the lectins exist as monomer, dimer, trimer and tetramer forms.
The present invention provides a diet and a dietary strategy to maximize the metabolic effects of lectin. In particular, soya fractions can be used such that the negative effects on the anti-nutritional fractions are reduced. Moreover, the beneficial effects of low doses of lectins can be used to enhance feed conversion of nutritionally poor soya fractions.